Mechanisms involved in the clearance of chylomicrons and aspects of the interactions at the vascular endothelium were studied in the rat. The poly-anion heparin, known to release lipoprotein lipase (LPL) from the vascular endothelium, enhanced the clearance of chylomicrons. Five minutes after heparin injection, the clearance of chylomicron triglycerides and retinyl esters was markedly accelerated. The rapid initial clearance was followed by a slower clearance of heavily lipolyzed chylomicrons. In contrast, one hour after heparin the clearance of both triglycerides and retinyl esters was retarded. This decreased removal of chylomicrons coincided with a decrease in the heparin releasable LPL activity, indicating that the previous release to plasma by heparin had resulted in net loss of functional LPL in the tissues. The poly-cation protamine released hepatic lipase and some LPL from their binding sites to plasma. One hour after protamine, plasma triglyceride levels were increased, indicating that chylomicron removal was impeded. It has been speculated that protamine inactivates LPL in vivo, but this was not the case. Ten minutes after injection of protamine normal amounts of LPL could be released by heparin. Thus, the accumulation of plasma triglycerides was not due to a rapid inactivation of LPL by protamine. LPL has specificity for sn-1,3-ester bonds. To investigate if this specificity is important in vivo, a lipid emulsion containing medium-chain fatty acids (MCFA) in the sn-1,3-position and long-chain fatty acids (LCFA) in the sn-2-position was synthesized, as well as an emulsion containing MCFA-TG mixed with LCFA-TGs (MMM/LLL). In vitro experiments showed large differences in the hydrolysis of the emulsions, but in vivo there were only small differences in the metabolism. To further study if lipid emulsions are cleared by the same mechanisms as chylomicrons, an emulsion was made by the same formulation as Intralipid® with addition of 3H-triolein and ,4C-cholesteryl ester. As measured by the removal of cholesteryl esters, the emulsion was cleared at the same rate as was chylomicrons. The triglyceride label was, however, removed more slowly from the emulsion droplets than from chylomicrons. Together with the lower recirculation of labeled free fatty acids (FFA) in plasma, this suggests that there was less lipolysis of the emulsion. The current view that removal of lipid emulsions in vivo is mainly dependent on LPL-mediated hydrolysis might thus not be correct. To further analyze the metabolism of chylomicrons, a compartmental model was developed. In this process, the distribution volume for chylomicrons was shown to be larger than the blood volume, a model for the metabolism of FFA in the rat was validated, and the full tissue distribution of injected chylomicrons was determined. According to the model, about half of the triglyceride label was removed from the circulation together with the core label while for the emulsion this number was about 80 %. In fasted rats all labeled fatty acids appeared to mix with the plasma FFA pool, while in fed rats about one-fifth of the fatty acids did not mix with the FFA but was apparently channeled directly to tissue metabolism. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1995, härtill 5 uppsatser.</p> / digitalisering@umu.se

Apis mellifera, otherwise known as the common honey bee, is an incredibly important social animal. Their important role in the world makes studying them of great importance. Their stomachs can be divided into three parts, the foregut, the midgut and the hindgut. The mouth and crop are located in the foregut, the midgut encompasses the ventriculus and the hindgut is made up of the ileum and rectum. Each part of a honey bee intestine hosts a different community of bacteria that vary in proportion with age, caste and season. These microbiota are essential for a honey bees mood, development and overall health. No two authors agree completely as to what the a honey bee’s gut microbiota is. In this thesis study the intestinal tract microbiome of four bee colonies, two of which belonging to the Apis mellifera carnica subspecies and two to the Apis mellifera buckfast subspecies, were sequenced. All four are from the same region in Sweden, Uddevalla. Many issues were found during this project, including one sick colony, but through them a more thorough and guaranteed method to sequence these honey bee intestinal bacteria was developed. The results of the sequencing showed that there is indeed a major difference in these intestinal communities even in bees from the same region or from the same subspecies. A possible culprit for the diseased colony was found.

Ribonucleotide reductase (RNR) is a key enzyme in the de novo biosynthesis and homeostatic maintenance of all four DNA building blocks by being able to make deoxyribonucleotides from the corresponding ribonucleotides. It is important for the cell to control the production of a balanced supply of the dNTPs to minimize misincorporations in DNA. Because RNR is the rate-limiting enzyme in DNA synthesis, it is an important target for antimicrobial and antiproliferative molecules. The enzyme RNR has one of the most sophisticated allosteric regulations known in Nature with four allosteric effectors (ATP, dATP, dGTP, and dTTP) and two allosteric sites. One of the sites (s-site) controls the substrate specificity of the enzyme, whereas the other one (a-site) regulates the overall activity.  The a-site binds either dATP, which inhibits the enzyme or ATP that activates the enzyme. In eukaryotes, ATP activation is directly through the a-site and in E. coli it is a cross-talk effect between the a and s-sites. It is important to study and get more knowledge about the overall activity regulation of RNR, both because it has an important physiological function, but also because it may provide important clues to the design of antibacterial and antiproliferative drugs, which can target RNR. Previous studies of class I RNRs, the class found in nearly all eukaryotes and many prokaryotes have revealed that the overall activity regulation is dependent on the formation of oligomeric complexes. The class I RNR consists of two subunits, a large α subunit, and a small β subunit. The oligomeric complexes vary between different species with the mammalian and yeast enzymes cycle between structurally different active and inactive α6β2 complexes, and the E. coli enzyme cycles between active α2β2 and inactive α4β4 complexes. Because RNR equilibrates between many different oligomeric forms that are not resolved by most conventional methods, we have used a technique termed gas-phase electrophoretic macromolecule analysis (GEMMA). In the present studies, our focus is on characterizing both prokaryotic and mammalian class I RNRs. In one of our projects, we have studied the class I RNR from Pseudomonas aeruginosa and found that it represents a novel mechanism of overall activity allosteric regulation, which is different from the two known overall activity allosteric regulation found in E. coli and eukaryotic RNRs, respectively.  The structural differences between the bacterial and the eukaryote class I RNRs are interesting from a drug developmental viewpoint because they open up the possibility of finding inhibitors that selectively target the pathogens. The biochemical data that we have published in the above project was later supported by crystal structure and solution X-ray scattering data that we published together with Derek T. Logan`s research group. We have also studied the effect of a novel antiproliferative molecule, NSC73735, on the oligomerization of the human RNR large subunit. This collaborative research results showed that the molecule NSC73735 is the first reported non-nucleoside molecule which alters the oligomerization to inhibit human RNR and the molecule disrupts the cell cycle distribution in human leukemia cells.

Zona pellucida glycoproteins (ZP) are important proteins for maturation of the oocytes in eukaryotes, these proteins are encoded by cluster of zp genes. zp2.3 and zp3.5 genes are expressed during the developing and maturation of the oocytes in zebrafish ovaries. Both of the gens have a CCAAT box in their promoter regions, playing a big role in the expression of the both genes in zebrafish oocytes. The transcription of the genes in the eukaryotes requires transcription factors to initiate and promote the transcription, the transcription factors can bind to the promoter region and initiate the transcription process. The nuclear factor y (NFY) regulates the genes by binding to the CCAAT boxes in their promoter regions, it consist from many subunit such as NF-YA and NF-YB. Here in this study we characterize the expression pattern of NF-YA and NF-YB by screening these genes expression in several organs and tissues, also to determine its roles in the expression of the zp2.3 and zp3.5 genes in the adultzebrafish ovary.

Numerical simulation of stochastic biochemical reaction networks has received much attention in the growing field of computational systems biology. Systems are frequently modeled as a continuous-time discrete space Markov chain, and the governing equation for the probability density of the system is the (chemical) master equation. The direct numerical solution of this equation suffers from an exponential growth in computational time and memory with the number of reacting species in the model. As a consequence, Monte Carlo simulation methods play an important role in the study of stochastic chemical networks. The stochastic simulation algorithm (SSA) due to Gillespie has been available for more than three decades, but due to the multi-scale property of the chemical systems and the slow convergence of Monte Carlo methods, much work is currently being done in order to devise more efficient approximate schemes. In this thesis we review recent work for the solution of the chemical master equation by direct methods, by exact Monte Carlo methods and by approximate and hybrid methods. We also describe two conceptually different numerical methods to reduce the computational time when studying models using the SSA. A hybrid method is proposed, which is based on the separation of species into two subsets based on the variance of the copy numbers. This method yields a significant speed-up when the system permits such a splitting of the state space. A different approach is taken in an algorithm that makes use of low-discrepancy sequences and the method of uniformization to reduce variance in the computed density function.

The chemical master equation (CME) describes the probability for the discrete molecular copy numbers that define the state of a chemical system. Each molecular species in the chemical model adds a dimension to the state space. The CME is a difference-differential equation which can be solved numerically if the state space is truncated at an upper limit of the copy number in each dimension. The size of the truncated CME suffers from an exponential growth for an increasing number of chemical species. In this thesis the chemical master equation is approximated by a continuous Fokker-Planck equation (FPE) which makes it possible to use sparser computational grids than for CME. FPE on conservative form is used to compute steady state solutions by computation of an extremal eigenvalue and the corresponding eigenvector as well as time-dependent solutions by an implicit time-stepping scheme. The performance of the numerical solution is compared to a standard Monte Carlo algorithm. The computational work for a solutions with the same estimated error is compared for the two methods. Depending on the problem, FPE or the Monte Carlo algorithm will be more efficient. FPE is well suited for problems in low dimensions, especially if high accuracy is desirable.

Ribosomes are macromolecular machines that are responsible for production of every protein in a living cell. Yet we do not know the details about how these machines are formed. The ribosome consists of four RNA strands and roughly 80 proteins that associate with each other in the nucleolus and form pre-ribosomal complexes. Eukaryotes, in contrast to prokaryotes, need more than 200 non-ribosomal factors to assemble ribosomes. These associate with pre-ribosomal complexes at different stages as they travel from the nucleolus to the cytoplasm and are required for pre-rRNA processing. We do however lack knowledge about the molecular function of most of these factors and what enables pre-rRNA processing. Especially, information is missing about how non-ribosomal factors influence folding of the pre-rRNA and to what extent the pre-ribosomal complexes are restructured during their maturation.  This thesis aims to obtain a better understanding of the earliest events of ribosome assembly, namely those that take place in the nucleolus. This has been achieved by studying the essential protein Mrd1 by mutational analysis in the yeast Saccharomyces cerevisiae as well as by obtaining structural information of nucleolar pre-ribosomal complexes. Mrd1 has a modular structure consisting of multiple RNA binding domains (RBDs) that we find is conserved throughout eukarya. We show that an evolutionary conserved linker region of Mrd1 is crucial for function of the protein and likely forms an essential module together with adjacent RBDs. By obtaining structural information of pre-ribosomal complexes at different stages, we elucidate what structuring events occur in the nucleolus.  We uncover a direct role of Mrd1 in structuring the pre-rRNA in early pre-ribosomal complexes, which provides an explanation for why pre-rRNA cannot be processed in Mrd1 mutants. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p>

Proteins have to be folded to their native structures to be functionally expressed. Misfolded proteins are proteotoxic and negatively impact on cellular fitness. To maintain the proteome functional proteins are under the constant surveillance of dedicated molecular chaperones that perform protein quality control (PQC). Using the model organism yeast Saccharomyces cerevisiae this thesis investigates the molecular mechanisms that cells employ to maintain protein homeostasis (proteostasis). In Study I the role of the molecular chaperone Hsp110 in the disentanglement and reactivation of aggregated proteins was investigated. We found that Hsp110 is essential for cellular protein disaggregation driven by the molecular chaperones Hsp40, Hsp70 and Hsp104 and characterized its involvement via regulation of Hsp70 ATPase activity as a nucleotide exchange factor. In Study II we found out that Hsp110 undergoes translational frameshifting during its expression resulting in a nuclear targeting. Nuclear Hsp110 interacts with Hsp70 and reprograms the proteostasis system to better deal with stress and to confer longevity. Study III describes regulation of Hsp70 function in PQC by the nucleotide exchange factor Fes1. We found that rare alternative splicing regulates Fes1 subcellular localization in the cytosol and nucleus and that the cytosolic isoform has a key role in PQC. In Study IV we have revealed the molecular mechanism that Fes1 employ in PQC. We show that Fes1 carries a specialized release domain (RD) that ensures the efficient release of protein substrates from Hsp70, explaining how Fes1 maintains the Hsp70-chaperone system clear of persistent misfolded proteins. In Study V we report on the use of a novel bioluminescent reporter (Nanoluc) for use in yeast to measure the gene expression and protein levels. In summary, this thesis contributes to the molecular understanding of chaperone-dependent PQC mechanisms both at the level of individual components as well as how they interact to ensure proteostasis. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 4: Manuscript.</p>

Alzheimer’s disease (AD) is a devastating neurodegenerative disease that affect millions of people worldwide. Aggregation of Amyloid-β (Aβ) monomers create toxic oligomers that can interact with cellular membranes and disturb cellular functions, resulting in cell death and neurological dysfunction. Increased levels of oxidative stress have been shown in the brains of AD patients, something that besides the obvious cell and tissue toxicity, also favors the amyloidogenic pathway and generates more Aβ monomers. Here we show that Edaravone, a free radical scavenger can affect the aggregation rate of different lengths of Aβ. We show that Aβ-40 that is more commonly found in vivo aggregates faster with addition of Edaravone, while Aβ-42 aggregates slower or not at all. These findings add up to previous findings where free radical scavengers and antioxidants such as Edaravone have been suggested as a potential treatment in Alzheimer’s disease.

AtPreP (Arabidopsis thaliana Presequence Protease) is a zink metallooligopeptidase that is dually targeted to both mitochondria and chloroplasts. In these organelles it functions as a peptidasome that degrades the N-terminal targeting peptides that are cleaved off from the mature protein after protein import, as well as other unstructured peptides. In A. thaliana there are two isoforms of PreP, AtPreP1 and AtPreP2.  We have performed characterization studies of single and double prep knockout plants. Immunoblot analysis revealed that both PreP isoforms are expressed in all tissues with highest expression levels in flowers and siliques. Furthermore, AtPreP1 was shown to be the most abundant isoform of the two. When comparing phenotype, the atprep2 mutant was similar to wild type, whereas the atprep1 mutant had a slight pale-green phenotype in the early developmental stages. The atprep1 atprep2 double knockout plants showed a chlorotic phenotype in true leaves, especially prominent during the early developmental stages. When analysing the first true leaves of double knockout plants, we found a significant decrease in chlorophyll a and b content. Mitochondrial respiratory rates measurements showed partially uncoupled mitochondria. Ultrastructure analysis using electron microscopy on double knockout plants showed aberrant chloroplasts with altered grana stacking and clearly fewer starch granules. Older plants showed less altered  phenotype, although there was a significant decrease in the accumulated biomass of about 40% compared to wild type. Peptidolytic activity studies showed no sign of compensatory mechanisms in the absence of AtPreP in mitochondria; in contrast we found a peptidolytic activity in the chloroplast membranes not related to AtPreP. In addition to zinc located in the catalytic site, crystallographic data revealed two Mg-binding sites in the AtPreP structure. To further investigate the role of these Mg-binding sites, we have made AtPreP variants that are unable to bind metal ions. Our data shows that one of these sites located close to the catalytic site is important for the activity of AtPreP. We also measured proteolytic activity of four human PreP-SNP variants and observed that the activity of all the hPreP-SNPs variants was lower; especially the hPreP-SNP (A525D) variant that displayed only 20-30 % of wild type activity. Interestingly, the activity was fully restored for all SNP-variants by addition of Mg2+.